Selection system for generating efficient packaging cells for lentiviral vectors

ABSTRACT

A method for selecting packaging cells that express high levels of gag/pol is provided.

This application claims benefit of 60/108,169, filed Nov. 13, 1998.

BACKGROUND OF THE INVENTION

Generation of efficient packaging cell lines for lentiviral vectors ishampered by the cytotoxicity of some of the products of the gag and polgenes. Thus, it is desirable to have inducible expression of gag and polso that optimal clones that will express gag and pol at high levels whenneeded can be selected in the absence of gag/pol expression.

SUMMARY OF THE INVENTION

A method for selecting cells which express gag and pol and thus areuseful as packaging cells is obtained by linking a selectable marker tothe gag/pol expression cassette of a packaging vector in such a way thatthe marker is expressed by the same promoter which controls expressionof the gag/pol genes although expression of the gag/pol genes issuppressed. Efficient expression of the marker predicts efficientexpression of the gag/pol genes on induction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a recombinant vector which exemplifies the instantinvention. The gag/pol sequences are flanked by splice donor and spliceacceptor sites. Also contained within the splice donor and spliceacceptor sites is an RRE (Rev responsive element).

FIG. 2 depicts the mechanism by which a vector containing an RRE wouldprovide inducible expression of only the marker gene, in the case of theexemplary recombinant vector of FIG. 1, the marker is CD4. In theabsence of Rev, splicing occurs between the splice donor and spliceacceptor sites thereby eliminating the gag/pol sequence. Only CD4 isexpressed. When Rev is present, splicing does not occur and the gag/polgenes are expressed.

FIG. 3 is a graph depicting the amounts of p24, a product of the gaggene, in culture medium when cells containing a vector of the instantinvention are propagated in the presence or absence of Rev. Twodifferent vectors were used, MDH spl CD4 and MD L g/p RRE. In bothvectors, the gag/pol genes are framed by splice donor and spliceacceptor sites and thus p24 is expressed when Rev is present in theculture.

DETAILED DESCRIPTION OF THE INVENTION

The invention takes advantage of the splicing control mechanisms of HIVand other lentiviruses which regulate expression of the late viralgenes, gag/pol and env, by means of a cis acting RNA element, RRE, and atrans acting regulatory protein, Rev. By the strategic placement ofsplice control elements, a switch in a gag/pol expression constructallows expression of a downs selectable marker gene in the basal stateand of the upstream gag/pol genes only on induction. As both genes aredriven by the same constitutive promoter, operation of the switch allowsfor gag/pol induction to an expression level related to that of theselectable marker.

Three features operate the switch: 1) the gal genes are contained withina splice donor site and one or more splice acceptor sites, wherein thesequences of the acceptor sites do not match the optimal consensussplice acceptor sequence (Lewin, “Genes”, John Wiley & Sons, NY)upstream of the marker gene; 2) the gag/pol genes contain sequenceswhich antagonize the expression of gag/pol (Schneider et al., J. Virol.71:4892-4903, 1997; Schwartz et al., J. Virol. 66:7176-7182, 1992); and3) the gag/pol genes are linked in cis to the RRE element as well asbeing separated from the Rev coding sequence.

A promoter which controls the expression of both gag/pol and the markergene is situated operably thereto, generally upstream from the gag/polsequences.

The RRE/Rev regulatory system is found in lentiviruses and thus, that ofHIV-1 or any other lentivirus can be used. Also, any other transcomplementing regulatory system which results in selective splicingwhich would control the expression of gag/pol as described herein can beused in the practice of the instant invention.

The first two features combine to suppress gag/pol expression in thebasal state. The third feature allows for Rev-dependent stimulation,i.e., induction, of the export of unspliced RNA and consequentexpression of the gag/pol genes.

Regarding the splice sites, a combination of an efficient splice donorsite, such as that of the 5′ major splice donor of HIV, and one or moresplice acceptor sites, wherein the splice acceptor sites do not matchexactly the optimal consensus, are used. Therefore, the splice acceptorsof interest are those an artisan would recognize as not being thatefficient, strong or good. Nevertheless, the splice acceptor sites areoperable, albeit at a suboptimal rate of efficiency. The suboptimalsplice sites appear to allow for more efficient expression from splicedtranscripts by the Rev-RRE system. An example of such a suboptimalsplice acceptor site is that of the third exon of the HIV-1 tat and revgenes.

Non-lentivirus splice donor and splice acceptor sites also can be usedin the practice of the instant invention so long as the splicing, andhence expression, of the gag/pol genes is controlled by the presence ofa trans acting factor, such as Rev.

The intrinsic instability of the lentiviral gag/pol coding sequences,and particularly the sequences contained in the intron, counteractsexpression in the basal state from unspliced transcripts that mayaccumulate due to the suboptimal nature of the splice sites. Anysequence which is known to be associated with the instability oftranscripts can be used in the practice of the instant invention.Instability sequences, however, such as those identified described inSchneider et al. and Schwartz et al., supra, in the gag/pol sequence,may not be strictly required for the operation of the switch.

Any of a number of possible selectable markers can be used. Markerswhich are readily detectable are desirable. For example, the marker maybe a cell surface molecule, which is antigenic, such as a CD molecule orlymphocyte antigen, or a light-emitting molecule, such as greenfluorescent protein. An artisan is free to select a selectable marker ofinterest from those known in the art.

The methods for cloning the various elements of the instant inventioninto a vector of interest are known in the art.

As a means of introducing yet another level of regulation, expression ofthe trans acting splice regulatory elements, in the case of HIV-1, Rev,can be inducible as well. In the presence of a separate inducible Revexpression construct, the expression of the gag/pol genes becomesinducible. For example, expression of Rev can be inducible using thetetracycline dependent regulatory system of Ory et al. (Proc. Natl.Acad. Sci. 93:11400-11406, 1996) wherein Rev is subcloned adjacent to atet operator. In the presence of tet, Rev is not expressed. However,when tet is withdrawn from the medium, Rev expression occurs.

Other known regulatory elements can be used as known in the art Thus, asuitable and known promoter can be place operably in the construct toregulate expression of the gag/pol and marker genes. Other regulatoryelements, such as a polyadenylation site can be used as desired.

Moreover, various modifications can be made to any one element includedin the vectors of interest to remove undesirable activities or toenhance desired activities. The artisan can rely on the known activitiesof the elements contemplated and can practice known techniques to effectthe desired changes, for example, deletion of sequences by selectivesubcloning, inactivation of a gene by site directed mutagenesis and soon.

An advantage of the instant invention is selection of optimal packagingclones for vectors, such as lentiviral-derived, and particularly,HIV-derived, vectors. Using a surface marker for the linked selection, apopulation of stable, high-level expressors can be sorted ontransfection of the constructs, and subsequently as often as needed tomaintain performance. In previously described linked-selection systems,expression of the marker gene is coupled to the expression of thedesired gene and cannot be operated in the reverse direction.

The instant method also can be used to select packaging clones forlentiviral vectors other than HIV-1, either using the HIV-1 Rev-RREsystem, or homologous elements of other lentivirus, so long as thehomologous regulatory elements functionally operate equivalently toyield selectable splicing of the gag/pol sequence in the presence of aninducer molecule located in trans to the coding sequences of interest.

The invention now will be exemplified in the following non-limitingexamples.

EXAMPLES

A packaging vector, pMDH L g/p RRE Spl CD4 (FIG. 1) was constructed toinclude the following: immediate/early enhancer/promoter of the humancytomegalovirus (CMV); HIV major 5′ splice donor, HIV gag/pol codingregions with optimised translation initiation sequence fitting the Kozakconsensus sequence (Dull et al. J. Virol. 72:8463-8471, 1998); HIV RREelement; HIV splice acceptor sites from the 3^(rd) exon of tat and rev;human CD4 coding region; and rat insulin poly-adenylation sequence.

The lentiviral packaging vector pMDH L g/p RRE Spl CD4 allows forselection of high level expression of the surface marker CD4 with verylow expression of the HIV-1 gag/pol genes. Due to the linkage of the CD4marker to the gag/pol genes, high expression of CD4 correlates with highinducible expression of gag/pol. In the absence of HIV Rev, splicing ofthe gag/pol sequences between the HIV splice donor and acceptors yieldefficient expression of CD4 without appreciable expression of gag andpol (FIG. 2A). In the presence of Rev, the RRE-mediated export ofunspliced gag/pol message allows expression of the gag pol proteins(FIG. 2B).

The pMDH L g/p RRE Spl CD4 plasmid was transfected into 293T (Dull etal., supra) with or without a Rev expression plasmid (Dull et al.,supra) and with a combination of other plasmids required to generatelentiviral vector delivery of a selectable marker, green fluorescentprotein (GFP).

About 4×10⁶293T cells were plated per 10 cm dish the night prior totransfection CaPO₄ co-transfection of the following plasmids wasperformed: pMDH L g/p RRE Spl CD4, 7 μg (HIV-derived gag/pol expressionplasmid); pRSV Rev, 2.5 μg; pCMV tat, 1 μg; pMD VSVG env, 3.5 μg; andpRRLhPGKGFPSIN-18, 10 μg (a self-inactivating HIV-derived transfervector carrying a green fluorescent protein coding sequence linked to aPGK promoter). Identical transfections also were performed without thepRSV Rev plasmid, and with the parental packaging vector pMD L g/p RREin place of pMDH L g/p RRE Spl CD4. Twenty hours after transfection,fresh medium was added and 24 hours later, conditioned medium washarvested for measuring the content of the HIV gag product, p24, byimmunocapture (Dupont) and for assaying transduction. The transfectedcells were harvested, incubated with phycoerythrin-labelled anti-CD4antibodies and analyzed by FACS for phycoerythrin and GFP fluorescence.

The transfectants were analyzed for expression of both CD4 and GFP, withand without HIV Rev. In both cases the vast majority of cells weredoubly positive for CD4 and GFP. As expected, the average expressionlevel of CD4 was higher in cells not expressing Rev. Expression of basallevels CD4 in the presence of Rev is due to the fact that Rev does notprevent completely the splicing of RRE-containing transcripts.

Similar transfections also were performed with the parental packagingvector pMD L g/p RRE in place of pMDH L g/p RRE Spl CD4. The vector pMDL g/p RRE expresses gag/pol of HIV in a Rev-dependent manner downstreamof a constitutively spliced intron derived from the β-globin gene. Cellsco-transfected with the pMD L g/p RRE packaging vector and Rev expressedgag/pol whereas in the absence of Rev, no gag/pol was detected.

Expression of the gag/pol genes in the transfected 293T cells wasanalyzed by measuring the content of the gag gene product, p24, in theconditioned medium by immunocapture (DuPont). FIG. 3 shows the p24concentration in the conditioned media of cells transfected with bothpackaging vectors in the presence and absence of HIV Rev. The Revdependence of gag/pol expression for both plasmid is evident. Theplasmid which contains the CD4 coding sequence expresses a very highlevel of p24 protein in the presence of Rev, similar to that obtainedwith the control plasmid.

Production of functional vector was analyzed by using the 293Tconditioned medium to transduce the GFP gene into HeLa cells. HeLa cellswere exposed to 10 μl of medium conditioned by cells transfected withthe pMDH L g/p RRE Spl CD4 packaging vector or the pMD L g/p RREpackaging vector in the presence (a) and absence (b) of HIV Rev.Transduction experiments were carried out by plating 5×10⁴ cells/well in6-well plates the night prior to infection. The next day, frozen 293Tconditioned medium was thawed and diluted 1:10, 1:100, 1:1000, and 1 mlof each dilution was used to infect the cells. Twenty hours afterinfection, fresh medium was added and 24 hours later, cells wereanalyzed by FACS for GFP expression.

The Rev-dependence of the transduction was evident for both plasmids.Only when Rev is expressed in vector producer cells do the target HeLacells express GFP. Moreover, the infectivity (transducing units/ng p24)of vector produced by either plasmid is similar, indicating that theCD4-linked plasmid operates as efficiently as the control plasmid.

We claim:
 1. A nucleic acid construct consisting of in operable linkagein the 5′ to 3′ direction; (1) a promoter; (2) a splice donor site; (3)a gag/pol coding sequence; (4) a Rev responsive element; (5) a spliceacceptor site; and (6) a selectable marker coding sequence.
 2. Thenucleic acid construct of claim 1, wherein the splice acceptor site isthat of the third exon of the HIV-1 tat and rev genes.
 3. The nucleicacid construct of claim 1, wherein the splice donor site is the major 5′splice donor site of HIV.
 4. A composition comprising: (a) a firstexpression cassette comprising the nucleic acid construct of claim 1;and (b) a second expression cassette comprising in operable linkage inthe 5′ to 3′ direction: (1) a promoter; and (2) a nucleic acid encodinga factor which binds to element (4) of said first expression cassette,which on such binding regulates splicing at said sites (2) and (5) ofsaid first expression cassette when an mRNA is transcribed from saidfirst expression cassette.
 5. The composition of claim 4, wherein thesplice acceptor suite is that of the third exon of the HIV-1 tat and revgenes.
 6. A composition comprising: (a) a first expression cassettecomprising in operable linkage in the 5′ to 3′ direction: (1) apromoter; (2) a splice donor site; (3) a gag/pol coding sequence; (4) aRev responsive element contiguous with; a splice acceptor site that isfrom the third exon of the HIV-1 tat and rev genes; and (5) a selectivemarker coding sequence; and (b) a second expression cassette comprisingin operable linkage in the 5′ to 3′ direction: (1) a promoter; and (2) anucleic acid encoding a factor which binds to element (4) of said firstexpression cassette, which on such binding regulates splicing at saidsites (2) and (4) of said first expression cassette when an mRNA istranscribed from said first expression cassette.
 7. The composition ofclaim 6, wherein the splice donor site of the first expression cassetteis the major 5′ splice donor site of HIV.